Interleukin-1 (IL-1) is important to the activation of T and B lymphocytes and mediates many inflammatory processes. cDNAs coding for two distinct forms of IL-1 have been isolated and expressed; these cDNAs represent two different gene products, termed I1-1.beta. (Auron, P. E., Webb, A. C., Rosenwasser, L. J., Mucci, S. F., Rich, A., Wolff, S. M. and Dinarello, C. A. [1984] Proc. Natl. Acad. Sci. USA 81:7909) and IL-1.alpha. (Lomedico, P. T., Gubler, U., Hellman, C. P., Dukovich, M., Giri, J. G., Pan, Y. E., Collier, K., Semionow, R., Chua, A. O. and Mizel. S. B. [1984] Nature 312:458). IL-1.beta. is the predominant form produced by human monocytes both at the mRNA and protein level. The two forms of human IL-1 share only 26% amino acid homology. Despite their distinct polypeptide sequences, the two forms of IL-1 have structural similarities (Auron, P. E., Rosenwasser, L. J., Matsushima, K., Copeland, T., Dinarello, C. A., Oppenheim, J. J. and Webb, A. C. [1985] J. Mol. Cell Immunol. 2:169), in that the amino acid homology is confined to discrete regions of the IL-1 molecule. The two forms of IL-1 also possess identical biological properties, including induction of fever, slow wave sleep, and neutrophilia, T- and B-lymphocyte activation, fibroblast proliferation, cytotoxicity for certain cells, induction of collagenases, synthesis of hepatic acute phase proteins, and increased production of colony stimulating factors and collagen. IL-1 also activates endothelial cells, resulting in increased leukocyte adhesiveness, PGI.sub.2 and PGE.sub.2 (prostaglandins) release, and synthesis of platelet activating factor, procoagulant activity, and a plasminogen activator inhibitor. Clearly, IL-1 plays a central role in local and systemic host responses. Because many of the biological effects of IL-1 are produced at picomolar (pg) concentrations in vivo, IL-1 production is likely a fundamental characteristic of host defense mechanisms.
In view of the multiple biological properties of IL-1 associated with inflammation and catabolic processes, the consequences of high levels of IL-1 in localized tissues such as bone and articular spaces are major concerns in the management and treatment of inflammatory diseases. Considerable attention has focused, therefore, on understanding the mechanisms of IL-1 production, and the precise function of IL-1 activity in inflammation. There are, then, two main areas of interest: measurement of IL-1 in body fluids and production of IL-1 from cells in vitro. At present, measurement of IL-1 produced in in vivo or in vitro is dependent on bioassays. T-cell assays for IL-1, based on the production of interleukin-2 (IL-2) or increased responsiveness to IL-2, are highly sensitive and can detect 10-100 pg/ml of IL-1 (Dinarello, C. A., Cannon, J. G., Mier, J. W., Bernheim, H. A., LoPreste, G., Lynn, D. L., Love, R. N., Webb, A. C., Auron, P. E., Reuben, R. C., Rich, A., Wolff, S. M. and Putney, S. D. [1986] J. Clin. Invest. 77:1734). However, these assays are vulnerable to interferences by several substances, including other lymphokines, such as IL-2 and IL-4 that directly stimulate T-cell division (Fox, D., Schlossman, S. and Reinherz, E. [1986] J. Immunol. 136:1945). The responses of endothelial (Bevilacqua, M. P., Pober, J. S., Majeau, G. R., Cotran, R. S. and Gimbrone, M. A. Jr. [1984] J. Exp. Med. 160:618; Dejana, E., Balconi, G., De Castellarnau, C., Barbieri, B., Vergara-Dauden, M. and de Gaetano, G. [1983] Biochim. Biophys. Acta 750:261) and synovial cells (Dayer, J-M, de Rochemonteix, Burrus, B., Demczuk, S. and Dinarello, C. A. [1986] J. Clin. Invest. 77:645) can be used to measure IL-1; some of these assays are adequately sensitive. But, these and other non-T-cell assays are not specific for IL-1 since the measured responses are also observed with other macrophage products, including tumor necrosis factor (Dinarello, C. A., Cannon, J. G., Wolff, S. M., Bernheim, H. A., Beutler, B., Cerami, A., Figari, I. S., Palladino, M. A. Jr. and O'Connor, J. V. [1986] J. Exp. Med. 163:1433; Beutler, B. and Cerami, A. [1986] Nature 320:584).
The measurement of IL-1 present in the supernates from cultured cells in vitro can be effected by the transfer into the IL-1 assays of substances used to either stimulate or suppress IL-1 production. For example, mitogens and adjuvants interfere with T-cell assays, while endotoxins and other bacterial products often mimic IL-1 activities in vivo. Several pharmacological agents which inhibit IL-1 production also interfere with biological assays for IL-1 (Dinarello, C. A., Marnoy, S. O. and Rosenwasser, L. J. [1983] J. Immunol. 130:890). Separating such agents from IL-1 can be difficult since most of these substances bind to protein. Prostaglandins and other arachidonic acid metabolites are also produced in cell cultures during stimulation of IL-1 production and their presence during IL-1 bioassays can either inhibit (Cahill, J. and Hopper, K. E. [1984] Int. J. Immunopharmacol. 6:9) or enhance (Kunkel, S. L. and Chensue, S. W. [1985] Biochem. Biophys. Res. Commun. 128:892) IL-1 activity. Monocytes themselves also produce polypeptide substances that have been shown to inhibit IL-1 activity in several assays (Arend, W. P., Joslin, F. G. and Massoni, R. J. [1985] J. Immunol. 134:3868). All of these factors show that the determination of IL-1 activity is not a straightforward process or a predictable process. Thus, there is a need in the art for a selective and predictable assay for determining the level of IL-1.beta. in human fluids, e.g., serum or urine.
The most widely employed IL-1 bioassay is the thymocyte co-stimulator or mouse lymphocyte activating factor (LAF) assay (Gery, I. and Waksman, B. H. [1972] J. Exp. Med. 136:143). The results of this assay are known to vary with the health and age of the mouse and the presence of thymic and accessory epithelial cells. Although the murine cloned T-helper cell line, D10.G4.1 exhibits remarkable sensitivity to IL-1 (Kaye, J., Gillis, S., Mizel, S. B., Shevach, E. M., Malek, T. R., Dinarello, C. A., Lachman, L. B. and Janeway, C. A. Jr. [1984] J. Immunol. 133:1339), these assays are also vulnerable to the presence of interfering substances "carried-over" from cell supernates or body fluids. Moreover, the response of these cells to IL-1 is often inconsistent between testing laboratories. The inconsistencies may be due to the requirement for macrophage feeder cells, growth factors and/or culture conditions. Some IL-1 T-cell assays involve a 2-step procedure whereby supernates are transferred to another cell line in order to measure IL-2 levels (Conlon, P .J. [1983] J. Immunol. 131:1280); this procedure is vulnerable to the presence of IL-2 in the original sample. The immunoassay described herein for IL-1.beta. offers the advantage, for the first time, of establishing standard methods for measuring IL-1.beta. and reducing the differences which occur because of different responding cells or individual laboratory practices.